Abstract
Cytochromes bc, key enzymes of respiration and photosynthesis, contain a highly conserved two-heme motif supporting cross-membrane electron transport (ET) that connects the two catalytic quinone-binding sites (Q(n) and Q(p)). Typically, this ET occurs from the low- to high-potential heme b, but in photosynthetic cytochrome b(6)f, the redox midpoint potentials (E(m)s) of these hemes remain uncertain. Our systematic redox titration analysis based on three independent and comprehensive low-temperature spectroscopies (continuous wave and pulse electron paramagnetic resonance (EPR) and optical spectroscopies) allowed for unambiguous assignment of spectral components of hemes in cytochrome b(6)f and revealed that E(m) of heme b(n) is unexpectedly low. Consequently, the cross-membrane ET occurs from the high- to low-potential heme introducing an uphill step in the energy landscape for the catalytic reaction. This slows down the ET through a low-potential chain, which can influence the mechanisms of reactions taking place at both Q(p) and Q(n) sites and modulate the efficiency of cyclic and linear ET in photosynthesis.